Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

- To begin, place five larvae in each lane of a six lane agarose plate. Add additional egg water, but don't overfill the lanes. Egg water contains methylene blue, which prevents mold growth. Let the larvae acclimatize for at least 10 minutes to reduce their stress.

Now place the well plate in an imaging chamber over a laptop screen. Focus the camera on the well plate. Fill the lane completely with egg water.

Next, record the behavior of the larvae placed on the blank background by taking time-lapse images. Display a moving red bar on the laptop screen, and take time lapse images of the larvae in response to the moving red bar.

The red shape is perceived as a threat, an aversive stimulus, and causes the larvae stress. In response, they avoid the bar and demonstrate positive thigmotaxis. Positive thigmotaxis is the fish's response to the stimulus seeking contact with the wall and swimming at the edge of the lane.

In the example protocol, we will analyze the avoidance and thigmotactic responses of zebrafish larvae treated with pharmaceutical compounds or environmental toxins.

- To begin image capture, carefully move the larvae from the Petri dish to the agarose lane to help reduce lar